VEGF is a family of proteins that were discovered on the basis of their ability to stimulate VEC (vascular endothelial cell) growth (angiogenesis). It now comprises five members, namely, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PLGF (placenta growth factor) that are encoded from distinct genes. Achen, et al., Proc. Nat'l. Acad. Sci. USA, 95: 548 (1998), Joukov, et al., EMBO J., 15: 1571 (1996), Maglione, et al., Oncogene, 8: 925 (1993), Olofsson, et al., Proc. Nat'l. Acad. Sci. USA, 93: 2576 (1996), Yamada, et al., Genomics, 42: 483 (1997). Each of the five members in turn comprises two or more isoforms that arise by the splicing of their respective pre-mRNAs. For example, the VEGF-A family includes VEGF206, VEGF189, VEGF183, VEGF165, VEGF145, VEGF121, and VEGF111. Anthony, et al., Placenta, 15: 557 (1994), Neufeld, et al., FASEB J., 13: 9 (1999), Lei, et al., Biochim. Biophys. Acta, 1443: 400 (1998), Jingjing, et al., Ophthamol. Vis. Sci., 40: 752 (1999), Cheung, et al., Am. J. Obstet. Gynecol., 173: 753 (1995), Burchardt, et al., Biol. Reprod., 60: 398 (1999). Among all VEGF proteins and isoforms, VEGF165 is by far the most frequently used form of VEGF both in basic and clinical studies.
It has been shown that, among different vascular cell types (endothelial, smooth muscle cells (SMC), and fibroblasts), SMC is the principal source for the secreted VEGF. Pueyo, et al., Exp. Cell Res., 238: 354 (1998). Expression of VEGF in SMC is upregulated by multiple factors including phorbol esters (Tischer, et al., J. Biol. Chem., 266: 11947 (1991)), cAMP (Claffey, et al., J. Biol. Chem., 267: 16317 (1992)), and hypoxia (Goldberg, et al., J. Biol. Chem., 269: 4355 (1994), Shweiki, et al., Proc. Nat'l Acad. Sci. USA, 92: 768 (1995)). The secreted VEGF acts on VEC principally through two different cell surface receptors, VEGFR-1 and VEGFR-2. Activation of VEGFR-1 results in VEC migration, while activation of VEGFR-2 VEC migration and proliferation. Waltenberger, et al., J. Biol. Chem., 269: 26988 (1994), Neufeld, et al., FASEB J., 13: 9 (1999), Ortega, et al., Front. Biosci., 4: D141 (1999). Although VEGFR-1 and VEGFR-2 have long been considered endothelium-specific, they have both been detected in human uterine and bovine aorta SMC. Grosskreutz, et al., Microvasc. Res., 58: 128 (1999), Brown, et al., Lab. Invest., 76: 254 (1997). Cultured uterine SMC responded to VEGF in the form of cell proliferation and cultured aorta SMC cell migration. Cultured human colon SMC, however, did not express VEGF receptors, nor did they respond to VEGF treatment. Brown, et al., Lab. Invest., 76: 254 (1997).
Angiogenesis is a complex process that includes activation, migration and proliferation of endothelial cells and formation of new blood vessels. D'Amore, et al., Ann. Rev. Physiol., 49(9-10): 453-64 (1987). VEGF has been shown to be intimately involved in the entire sequence of events leading to growth of new blood vessels. Gross, et al., Proc. Nat'l. Acad. Sci., 80(9): 2623-27 (1983), Folkman, et al., Proc. Nat'l. Acad. Sci., 76(10): 5217-21 (1979). Five human VEGF isoforms of 121, 145, 165, 189 and 206 amino acids have been isolated. Gross, et al., Proc. Nat'l. Acad. Sci., 80(9): 2623-27 (1983), Leung, et al., Science, 246: 1306-09 (1989), Poltorak, et al., J. Biol. Chem., 272(11): 7151-78 (1997). Among the isoforms, VEGF 165 seems to be the most effective and most commonly used. The effect of VEGF 165 in augmenting perfusion and in stimulating formation of collateral vessels has been shown in animal models Hopkins, et al., J. Vascular Surgery, 27(5): 886-94 (1998), Asahara, et al., Circulation, 91(11): 2793-801 (1995), Hariawala, et al., J. Surg. Res., 63(1): 77-82 (1996), Bauters, et al., Circulation, 91(11): 2802-9 (1995), Bauters, C., et al., Am. J. Physiol., 267(4 Pt 2): H1263-71 (1994), Takeshita, et al., J. Clin. Invest., 93(2): 662-70 (1994), Takeshita, et al., Circulation, 90(5 Pt 2): II228-34 (1994), Takeshita, et al., Am. J. Path., 147(6): 1649-60 (1995), Banai, et al., Circulation, 89(5): 2183-9 (1994). In clinical trials, successful induction of collateral blood vessels in ischemic heart disease and critical limb ischemia by VEGF have also been reported. Baumgartner, et al., Circulation, 97(12): 1114-23 (1998), Losordo, et al., Am. Heart J., 138(2 Pt 2): 132-41 (1999).
Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin, stimulating both connective tissues and neuroglial cells. PDGF also acts as a potent chemoattractant for mesenchymal cells, mononuclear cells, and neutrophils. PDGF is stored in platelet granules and released with platelet activation. Other cell types also produce PDGF, including endothelial cells, monocytes/macrophages, vascular smooth muscle cells, fibroblasts, and cytotrophoblasts. PDGF consists of disulfide-linked dimers of αα, αβ, or ββ configuration. PDGF has a short half-life and usually produces only local effects. Two distinct PDGF receptors have been identified that are structurally related and have an intracellular protein kinase domain.
Neurotrophins are a class of structurally related growth factors that promote neural survival and differentiation. They stimulate neurite outgrowth, suggesting that they can promote regeneration of injured neurons, and act as target-derived neurotrophic factors to stimulate collateral sprouting in target tissues that produce the neurotrophin. Korsching, J. Neurosci., 13: 2739 (1993). Recently, local synthesis and autocrine mechanisms of action have been reported. Lewin and Barde, Ann. Rev. Neurosci., 19: 289 (1996). In vivo overexpression of a neurotrophic factor, through gene transfer, would ensure local and continuous neurotrophin production in a manner resembling the physiologic, as these proteins are usually produced and secreted by target and glial cells surrounding neurons.
Neurotrophin-3 (NT-3) is a member of the neurotrophin class of structurally related growth factors. NT-3 is a 27 kDa homodimer that supports the growth and survival of sympathetic neurons as well as sensory neurons. NT-3 is highly conserved across species and is primarily expressed in kidney, spleen, and heart with lower expression levels found in the skin, skeletal muscle, lung, thymus, and ovaries. NT-3 binds the low affinity NGF receptor, p75NTR, and may initiate apoptosis through this receptor. NT-3 also binds and induces signaling through the TrkC receptor.
Neurotrophin-4 (NT-4) is yet another member of this class of neurotrophins. NT-4 is a homodimer that supports the growth and survival of sympathetic neurons, dorsal root ganglion neurons, nodose ganglion neurons, basal forebrain cholinergic neurons and neurons of the locus coeruleus. NT-4 is less highly conserved between species, unlike other neurotrophins. NT-4 expression is widespread in brain and peripheral tissues. NT-4 induces cellular signaling through the p75NTR receptor as well as the TrkB receptor.
Brain-derived neurotrophic factor (BDNF), another member of the neurotrophins, was initially characterized as a basic protein present in brain extracts and capable of increasing the survival of dorsal root ganglia. Leibrock, et al., Nature, 341: 149 (1989). When axonal communication with the cell body is interrupted by injury, Schwann cells produce neurotrophic factors such as nerve growth factor (NGF) and BDNF. Neurotrophins are released from the Schwann cells and dispersed diffusely in gradient fashion around regenerating axons, which then extend distally along the neurotrophins' density gradient. Ide, Neurosci. Res., 25: 101 (1996). Local application of BDNF to transected nerves in neonatal rats has been shown to prevent the massive death of motor neurons that follows axotomy. DiStefano, et al., Neuron, 8:983 (1992), Oppenheim, et al., Nature, 360: 755 (1992), Yan, et al., Nature, 360: 753 (1992). The mRNA titer of BDNF increases to several times the normal level 4 days after axotomy and reaches its maximum at 4 weeks. Meyer, et al., J. Cell Biol., 119: 45 (1992). Moreover, BDNF has been reported to enhance the survival of cholinergic neurons in culture. Nonomura, et al., Brain Res., 683: 129 (1995).
Angiopoietin-1 (Ang-1) is a member of a family of endothelium growth factors. Ang-1 is a ligand for the Tie-2 receptor, a receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains expressed primarily on endothelial cells and very early hematopoietic cells. Ang-1 promotes chemotaxis, cell survival, cell sprouting, vessel growth and stabilization of Tie-2-expressing endothelial cells. Ang-1 is thought to have a distinct angiogenic role from that of VEGF involving the recruitment of peri-endothelial cells that will become pericytes and smooth muscle tissue of the blood vessel, thereby maintaining the stability of the blood vessels. See, e.g., Hanahan, D., Science, 277: 48-50.
Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factor family. bFGF stimulates the proliferation of all cells of mesodermal origin including smooth muscle cells, neuroblasts, and endothelial cells. bFGF stimulates neuron differentiation, survival, and regeneration. In vitro functions suggest that bFGF modulates angiogenesis, wound healing and tissue repair, and neuronal function in vivo. bFGF, a heparin-binding growth factor, is capable of inducing functionally significant angiogenesis in models of myocardial and limb ischemia. Zheng, et al., Am. J. Physiol. Heart Circ. Physiol., 280: H909-17 (2001), Laham, et al., J. Am. Coll. Cardiol., 36: 2132-39 (2000), Laham, et al., Curr. Interv. Cardiol. Rep., 1: 228 (1999), Unger, et al., Am. J. Cardiol., 85: 1414-19 (2000), Kawasuji, et al., Ann. Thorac. Surg., 69: 1155 (2000), Rajanayagam, et al., J. Am. Coll. Cardiol., 35: 519 (2000), Kornowski, et al., Circulation, 101: 545-48 (2000), Ohara, et al., Gene Ther., 8: 837 (2001), Lazarous, et al., J. Am. Coll. Cardiol., 36: 1239 (2000), Rakue, et al., Japan Circ. J., 62: 933-39 (1998), Baffour, et al., J. Vasc. Surg., 16: 181 (1992).
Erectile function is a hemodynamic process of blood in-flow and pressure maintenance in the cavernosal spaces. Christ, Urol. Clin. North Am., 22: 727 (1995). Following sexual arousal and the release of nitric oxide to the erectile tissue, three processes occur to achieve an erection. These are relaxation of the trabecular smooth muscle, arterial dilation and venous compression. Id. During this final stage, arterial flow fills sinusoidal spaces, compressing subtunical venules thereby reducing venous outflow. Blood flows into the cavernous spaces of the penis, thus expanding and stretching the penis into a rigid organ. The flow of blood in and out of the cavernous spaces is controlled by cavernous smooth muscle cells (CSMC) embedded in the trabeculae of the cavernous spaces. With normal erectile function, a high intracavernous pressure (ICP) is maintained with a low inflow rate. Karadeniz, et al., Urol. Int., 57: 85 (1996).
As such, the penis is a predominantly vascular organ, and vascular or penile arterial insufficiency is the most common etiology of erectile dysfunction (ED). Sinusoidal smooth muscle atrophy and collagen deposition is a common finding in men with long standing ED of various etiologies, whether due to hormonal, neurological or vascular causes. Karadeniz, et al., Urol. Int., 57: 58 (1996). Such degradation in smooth muscle quantity and quality leads to veno-occlusive dysfunction. This represents an end-stage muscular degeneration akin to myocardial changes with congestive heart failure or dilated cardiomyopathy for which no treatment currently exists with hope of reversing the underlying pathologic process.
Veno-occlusive disease is a common finding among patients with erectile dysfunction (ED). Following radical prostatectomy, for example, approximately 30% of patients may have vasculogenic ED in addition to neurogenic ED and at least half of these men may have venous leak. Regardless of the etiology of organic ED (neurogenic, traumatic, hormonal, and vascular, etc.), venous leakage is a common final condition resulting from smooth muscle atrophy. Mersdorf, et al., J. Urol., 154: 749 (1991). Veno-occlusive dysfunction is the most common etiology of ED among non-responders to medical management of ED. None of the medical therapy currently exists is curative for this condition. Patients with veno-occlusive dysfunction exhibit a poor response to intracavernous injection with vasoactive agents (papavarine, prostaglandin E1, phentolamine, or combinations, for example), despite good arterial flow demonstrated by duplex ultrasound. The diagnosis of veno-occlusive disease may be confirmed with specific findings on cavemosometry and cavemosography. Nehra, et al., J. Urol., 156: 1320 (1996).
Atherosclerotic or traumatic arterial occlusive disease of the pudendal-cavernous-helicine arterial tree can decrease the perfusion pressure and arterial flow to the sinusoidal spaces, thus decreasing the rigidity of the erect penis. Common risk factors associated with generalized arterial insufficiency include hypertension, hyperlipidemia, cigarette smoking, diabetes mellitus, and pelvic irradiation. Goldstein, et al., JAMA, 251: 903-910 (1984), Rosen, M. P., et al., Radiology, 174(3 Pt 2): 1043-48 (1990), Levine, F. J., et al., J. Urology, 144(5): 1147-53 (1990). Epidemiological studies have shown a high incidence of ED in patients with coronary arterial disease. Heaton, J. P., et al., Int'l J. Impotence Res., 8(1): 35-39 (1996). Focal lesion of the common penile or cavernous artery is most often seen in young patients who have sustained blunt pelvic or perineal trauma such as in cases of biking accidents. Levine, F. J., et al., J. Urology, 144(5): 1147-53 (1990).
Because of the close proximity of the cavernous nerves to the capsule of the prostate, ED is a frequent complication after radical prostatectomy or cystectomy and prostatic cryosurgery. Although the nerve-sparing prostatectomy technique developed by Walsh, et al., Br. J. Urol., 56: 694 (1984) has significantly reduced the postoperative impotence rate, a large number of patients still suffer from inadequate penile rigidity. Peripheral nerve regeneration is a slow process, and the fact that most patients do not recover potency for 6 months to 2 years indicates substantial axonal damage, even with preservation of the neural sheath. An anatomic study of the cavernous nerves by Paick et al., Urology, 42: 145 (1993) revealed both a medial and a lateral bundle of cavernous nerves at the level of the prostate, suggesting that in some cases the lateral bundle can be saved, even in non-nerve-sparing prostatectomy.
The sprouting of the remaining nerves in penile tissue appears to be more important in regeneration than re-growth of nerves through the damaged and fibrotic tissues. The importance of sprouting in the remaining nerves was confirmed in an animal study that revealed regeneration of the cavernous nerves after unilateral resection. Carrier, et al., J. Urol., 153: 1722 (1995). In addition, a previous study in our laboratory showed that systemic growth hormone injection significantly enhanced cavernous nerve regeneration after unilateral injury. Jung, et al., J. Urol., 160: 1899 (1998).
Methods for treating erectile dysfunction have included from the administration of prostaglandin E (U.S. Pat. No. 5,942,545), local administration of vascular muscle relaxants and vasoactive pharmaceutical agents. See, for example, U.S. Pat. Nos. 5,942,545, 6,056,966; and 5,646,181.
Advancement in molecular biology has brought improved understanding of pathophysiology on the gene and molecular level, and offers promise of treatment possibilities aimed at a specific pathologic molecular mechanism. As in other vasculopathies such as limb claudication (Baumgartner, et al., Circulation, 97: 1114 (1998) and coronary artery disease (Symes, et al., Ann. Thorac. Surg., 68: 830 (1999), treatment with VEGF in either protein or gene form has increased neovascularity in animal models and improved symptomatic angina and wound healing in humans with inoperable heart disease and critical limb ischemia, respectively. The penis represents a convenient tissue target for gene or growth factor therapy due to the penis' external location on the body, ubiquity of endothelial-lined spaces and low-level blood flow in the flaccid state. In addition, the penis is filled with billions of endothelial and smooth muscle cells both are rich in VEGF receptors. Liu, et al., J. Urol., 166: 354-360 (2001).
Recently, we have established an animal model in which CSMC was seen decreased following internal iliac artery ligation that restricted blood supply to the penis. However, rats treated with intracavernous injection of vascular endothelial growth factor (VEGF) shortly after internal iliac artery ligation had nearly normal CSMC. The protective effects of VEGF on CSMC could be due to partial restoration of blood supply as VEGF is expected to stimulate vascular endothelial cell (VEC) proliferation. Lin, et al., Proc. Nat'l Acad. Sci. USA, 97: 10242-47 (2000). Alternatively, VEGF might act directly on CSMC, as we will present evidence that CSMC express one of the two principal VEGF receptors. Sondell, et al., Eur. J. Neurosci., 12: 4243-54 (2000); Liu, et al., J. Urol., 166: 354-360 (2001).
Females can also have sexual dysfunction, and this dysfunction can increase with age. It is usually associated with the presence of vascular risk factors, genital smooth muscle atrophy, and onset of menopause. Some of the vascular and muscular mechanisms that contribute to penile erection in the male are believed to be similar vasculogenic factors in the female genital response. It is known that in women sexual arousal is accompanied by arterial inflow which engorges the vagina and increases vaginal lubrication, and that the muscles in the clitoris and the perineum assist in achieving clitoral erection.
In the female patient, sexual arousal disorder can arise from organic and pyschogenic causes, or from a combination of the foregoing. Female sexual arousal disorder is classified into five categories: 1) hypoactive sexual desire disorder, 2) sexual aversion disorder, 3) sexual arousal disorder, 4) orgasmic disorder, and 5) sexual pain disorder. The present invention applies to sexual arousal disorder. Sexual arousal disorder is the persistent or recurring inability to attain or maintain adequate sexual excitement, causing personal distress. It may be experienced as the lack of subjective excitement or the lack of genital lubrication or swelling or other somatic responses. Organic female sexual arousal disorder is known to be related in part to vasculogenic impairment resulting in inadequate blood flow, vaginal engorgement insufficiency and clitorial erection insufficiency. Animal studies have demonstrated the dependence of vaginal vascular engorgement and clitoral erection on blood flow. See, for example, Park et al., “Vasculogenic female sexual dysfunction: the hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency, “Int'l J. Impotence Res., 9(1), 27-37 (March 1997).
Female sexual dysfunction has been treated with pharmacological intervention to stimulate blood flow as well as with prostaglandins. See, for example, U.S. Pat. Nos. 6,193,992 B1; 5,945,117; 6,031,002; and 5,891,915.